The 3D structure was completed by Phyre2 software, using the structure of an scFv antibody in complex with an analogue of the main immunogenic region of the acetylcholine receptor (PDB code: 1F3R) as templates. for the generation of a rabbit recombinant monoclonal antibody for specific detection and monitoring of nitrogen-fixing bacteria in both free-living form and in plant nodules. sp. DOA9, a broad host range soil bacteria, originally isolated from the root nodules of in Thailand was used as a model in this study. A recombinant single-chain fragment variable (scFv) antibody library was constructed from the spleen of a rabbit immunized with DOA9. After three rounds of biopanning, one specific phage-displayed scFv antibody, designated bDOA9rb8, was identified. Specific binding of this antibody was confirmed by phage enzyme-linked immunosorbent assay (phage ELISA). The phage antibody could bind specifically to DOA9 in both free-living cells (pure culture) and bacteroids inside plant nodules. In addition to phage ELISA, specific and robust immunofluorescence staining of both free-living and bacteroid forms could also CCND3 be observed by confocal-immunofluorescence imaging, without cross-reactivity with other tested bradyrhizobial strains. Moreover, specific binding of free scFv to DOA9 was also demonstrated by ELISA. This recombinant antibody can also be used for the study of the molecular mechanism Edasalonexent of plantCmicrobe interactions in the future. Introduction is one of the soil bacteria that can fix nitrogen in symbiosis with specific legumes and convert nitrogen gas into ammonia to be used as fertilizer in plants. sp. DOA9 was originally isolated from the root nodules of in Thailand. This strain was classified as based on multilocus DNA sequence analysis of 16S rRNA and housekeeping genes (can be done by many methods including microbial, serology, and DNA techniques [3]. Serology is a simple and rapid technique when compared to microbiological or DNA-based techniques [4]. Both polyclonal and monoclonal antibodies have been used for Edasalonexent enzyme-linked immunosorbent assay (ELISA), immunoagglutination, or immunofluorescence detection of target strains in culture broth or in nodules, as well as for monitoring the persistence of inoculant in the soil [5C7]. Serological technique can also be used as a tool to study the key determinants involved in legume-rhizobium nodulation. Monoclonal antibodies were used as analytical probes to elucidate the structural differences among lipopolysaccharide (LPS) molecules in strain 3841 in comparison with LPS-defective mutant derivatives [8]. Polyclonal antibodies raised against Edasalonexent LPS from bradyrhizobial strains that nodulate peanut have been shown to cross-react with the peanut specific bradyrhizobia but not [9]. This result suggested the presence of certain common LPS antigenic determinants among the strains. Therefore, it is possible that polyclonal antibody could have cross-reactivity with other indigenous bacteria sharing similar antigenic determinant presence in the soil, creating difficulty in result interpretation [5]. Application of monoclonal antibodies for identification of a rhizobial strain has been done for the first time against a commercial inoculant of 162X95 [9]. This monoclonal antibody was used in an indirect ELISA to differentiate strain 162X95 from indigenous strains collected from the Appalachian region where this strain was isolated, and also used for detecting the bacteroid from crushed nodules with a strong immunological reaction. However, the traditional method for monoclonal production is quite complicated. Moreover, monoclonal antibodies may not be the ideal method for bacterial identification if those monoclonal antibodies bind with nonspecific or invariant antigenic determinant molecule among bacteria. Therefore, the efficient method of production and screening the monoclonal antibody that bind with the specific antigenic molecule of each rhizobium are required. The development of antibody engineering techniques has provided ways to produce antibodies in many formats [10]. Compared to all antibody-based methods, phage-display technology is a highly attractive alternative method for the production of monoclonal antibodies against diverse antigens [11]. The phage-display technique allows isolation of antibodies directly.